Zeaxanthin (3,3'-dihydroxy-.beta.-carotene) is a carotenoid that imparts the yellow color to corn, egg yolks and the skin of poultry. It can be used as a feed additive and as a colorant in the cosmetic and food industries.
The pure zeaxanthin and the pigment-containing cell-mass manufactured according to the process of this invention can be used for the coloring of foodstuffs, as well as for the coloring of cosmetic preparations. The pigment-containing cell-mass is particularly suitable for the coloring of legs, beaks, skin, fat, flesh and egg-yolk of poultry.
Zeaxanthin is synthesized biologically by very few bacterial species of the genus Flavobacterium. With rare exceptions, all Flavobacterium species may be conveniently placed into two categories: 1) strongly proteolytic (digestion of gelatin, casein, and coagulated serum), and 2) nonproteolytic. F. meningosepticum (biovar IIa) and F. indologenes (biovar IIb) are always proteolytic; other flavobacteria are not (IIc, IIe, IIh, IIi, and IIk-2). F, multivorum is one of three species now recognized in CDC's IIk-2 group (non-proteolytic).
For poultry producers, there has been a long history of problems with stability and biological availability, particularly with xanthophylls from marigolds and alfalfa. Much work goes on to measure and improve these properties. Most marigold products must be solvent-extracted and saponified, and may require the inclusion of antioxidants in the extraction process (Marusich and Bauerfeind, Carotenoids As Colorants and Vitamin A Precursors; ed., J. C. Bauerfeind, Academic Press, 1981). The feeding studies shown in Example 6 directly compared a known amount of pigment versus several processed extracts from marigolds; the data indicate that the composition of the present invention is biologically available and stable, pigments faster, and is 2-3 times more potent, on a pure pigment basis, than marigold xanthophylls.
It is known that several species of Flavobacterium, under certain process conditions and/or using certain nutrient media, are capable of producing zeaxanthin (see the Relevant Literature section).
It is also known that improved yields of zeaxanthin may be obtained by culturing a microorganism of the genus Flavobacterium under conditions whereby the amounts of carbon and nitrogen present in the culture medium are maintained at a substantially constant ratio. The nutrient media of these processes require glucose and other nutrients, which are relatively costly and require a long culturing period.
On the other hand, the present invention provides a process for preparing zeaxanthin by culturing a zeaxanthin-producing microorganism of Flavobacterium multivorum, the first time that this species of Flavobacterium has been shown to produce pigment. Moreover, the nutrient medium in which the microorganism is cultured is a relatively low cost nutrient medium. Furthermore, the culturing period is much faster and more efficient than known methods.
Accordingly, an object of the present invention is to provide a process for the preparation of zeaxanthin using a strain of Flavobacterium multivorum, or a mutant or variant thereof. The process according to the present invention makes it possible to obtain increased amounts of zeaxanthin and increased cell yields of zeaxanthin-containing cells in comparison to known microorganisms and processes.
It is another object to provide a process for producing increased yields of the biologically available zeaxanthin pigment.
It is another object to provide a process for producing zeaxanthin using microorganisms that are non-fastidious, grow rapidly, and are non-pathogenic. These microorganisms are also capable of using a nutrient medium containing many different carbon sources.
It is another object to provide an economical and commercially feasible nutrient medium, which optimizes the amount of zeaxanthin produced, provides a high cell yield, provides a high yield of zeaxanthin, and decreases the fermentation period for producing zeaxanthin.
Another object is to provide a bacterial species that produces zeaxanthin.
Another object is to provide a microorganism heretofore not known to produce zeaxanthin.
These objects and others will become apparent in the following description of the invention and in the claims.